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Transformation Nonresponsive Cells Owe Their Resistance to Lack of p65/Nuclear Factor-B Activation¹

Frederick Cancer Research and Development Center [R. N., P. T., C. E. C., G. A. H., M. R. Y., N. H. C.], National Cancer Institute and IRSP, Science Applications International Corporation-Frederick, Frederick, Maryland 21702 [T-C. H.]

Clonal variants of mouse epidermal JB6 cells that are genetically susceptible (P+) or resistant (P-) to tumor promoter-induced neoplastic transformation exhibit differential activator protein-1 (AP-1) response. Transactivation of AP-1 appears to be necessary but not sufficient to promote transformation in JB6 cells. Inhibition of AP-1 is invariably accompanied by inhibition of nuclear factor-B (NF-B) when transformation is suppressed, suggesting that NF-B may also play a role in neoplastic transformation. We report here that transactivation of NF-B is inducible by tumor promoters in P+ but not in P- JB6 cells. Inhibition of NF-B using a nondegradable mutant of IB suppressed inducible anchorage-independent transformation of P+ JB6 cells, suggesting that NF-B activation is required for tumor promotion. Induced degradation of IB occurred in both P+ and P- JB6 cells, indicating that failure to activate NF-B in P- JB6 cells cannot be attributed to failure to degrade IB. Slightly higher levels of nuclear p65 were seen in P+ than in P- JB6 cells. The p65-specific DNA binding activity was also higher in P+ cells upon induction by tumor necrosis factor-, suggesting that differential NF-B activation may be attributable to changes in p65 activity. Transactivation of p65 protein was substantially higher in P+ than in P- JB6 cells, as determined by assay of Gal4-p65 fusion constructs. Thus activated, p65 may be a limiting factor for NF-B activation and transformation responses. Stable expression of p65 in P- JB6 cells conferred not only inducible NF-B and AP-1 activation but also transformation response to tumor promoters. Therefore, p65/NF-B appears to be not only necessary for but also sufficient to confer tumor promotion response. Although stable expression of p65 in P- cells produced p65 increases in whole cell extracts, only the transfectants exhibiting increased nuclear p65 showed transformation response. Thus, elevation of nuclear p65 appears to be a necessary step for a transformation response. The P-/p65 transfectants showing acquired transformation response also showed elevated p65-specific transactivation response, thus recapitulating the NF-B phenotypes seen in P+ cells. Expression of a transactivation-deficient mutant of Jun or dominant-negative extracellular signal-regulated kinase suppressed both AP-1 activation and p65-specific transactivation in JB6 cells, suggesting that AP-1 activity is needed for p65 transactivation and consequently for NF-B activation. Thus, the transformation nonresponsive P- JB6 cells owe their resistance to lack of NF-B activation and p65 transactivation that appears in turn to be attributable to insufficient AP-1 activation.

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